The long-term objective of this project is the development of a safe and highly efficacious live oral cholera vaccine. Previous work funded by this project has resulted in the development of an attenuated live oral cholera vaccine, Vibrio cholerae CVD 103-HgR, which confers strong protective immunity after a single dose. Although this vaccine is highly protective against experimental challenge in North Americans, it has a mixed record of success in conferring protective immunity in citizens of developing countries. An improved cholera vaccine that colonizes the intestine more avidly and engenders a stronger immune response than CVD 103-HgR would be desirable. However, better colonizing and more immunogenic vaccine strains have usually caused side effects in North Americans including mild to moderate diarrhea. During the past period of support, we have used in vivo expression technology (IVET) in a volunteer study to identify genes that are activated during the course of human infection. We have also generated new insights into intestinal inflammation caused by these reactogenic cholera vaccines by using gene arrays of epithelial cell transcriptional responses. In the next period of support, we will pursue four specific aims. The first aim will be to examine the role of flagellin in TLR5 activation and IL-8 production by V. cholerae. The second aim will be to characterize the involvement of TLR5 in the reactogenicity of many live attenuated cholera vaccines. The third aim will be to characterize the human immune response to in vivo-induced V. cholerae proteins identified in the IVET volunteer study. The fourth aim will be to develop a V. cholerae vector system for expression of foreign antigens based on in vivo induced promoters. The proposed aims will yield important information about the interaction of V. cholerae with the innate immune system, insights into the reactogenicity caused by ctx V. cholerae mutants, novel information regarding human immune responses to in vivo expressed antigens, and a promising new vector system for expression of heterologous antigens. Lay summary: Vibrio cholerae is a bacterium that causes cholera, a severe diarrheal disease that can often cause death. This project will develop improved vaccines for the prevention of cholera.